Chemical analysis of aqueous fluids, such as effluent streams, and biological fluids are important for health maintenance and diagnostic care. Frequently, the material to be determined, the analyte, is present in the fluids in very small amounts and conventional analytical methods lack the sensitivity to accurately measure such small quantities.
Many biologically significant substances can be quantifiably determined using a reaction or sequence of reactions that produce a reductant. For example, nicotinamide adenine dinucleotide, reduced form, otherwise known as NADH, is commonly produced in these reactions. Another common reductant is the corresponding phosphate, otherwise known as NADPH. Conventional assay methods usually monitor NADH or NADPH by directly measuring the change in the solution absorbance when these species are produced. These conventional assays suffer from the drawbacks that they are relatively insensitive and are limited to measurements at about 340 nm, where many interferents also have absorptions.
To improve the sensitivity of these assays, these species are used to reduce a tetrazolium salt to yield a formazan dye. However, formazan dyes themselves have low extinction coefficients and thus, the sensitivity is not sufficiently improved by this method.
It has also been suggested in U.S. Pat. No. 3,331,752 to reduce a ferric ion chelate with the NADH to produce a ferrous ion chelate with different absorption characteristics than the ferric ion chelate. This method also has less than the desired sensitivity since there is no amplification step involved.
In copending commonly assigned U.S. Pat. No. 4,701,420, issued Oct. 20, 1987, entitled ANALYTICAL COMPOSITIONS, ELEMENTS AND METHODS UTILIZING REDUCTION OF FERRIC ION CHELATES TO FORM DETECTABLE DYES, there is disclosed an improved method involving ferric ion chelates. While this method involves advantages over that disclosed in the '752 patent, it still does not provide an amplification step and therefore does not have the required sensitivity.
The problem to be solved, therefore, is to provide methods and reagent compositions with improved sensitivity. This is accomplished by providing an amplification step. Amplification occurs when one equivalent of an analyte gives more than one equivalent of detectable species.